Our studies during the previous funding period focused on understanding the relationship between hypoxia and stem like breast cancer cell (SBCC) markers such as CD44, and understanding the effect of targeting hypoxia and choline metabolism on their expression. These studies identified hypoxia as a regulator of CD44. Hypoxia inducible factor (HIF) silencing reduced, but did not eliminate, CD44 expression in primary and metastatic tumors, and decreased metastasis from these tumors in a CD44 expression dependent manner. In human breast cancers, we observed strong expression of CD44 in breast cancer cells and in cancer associated fibroblasts (CAFs) that were closely associated with straight long collagen 1 (Col1) fibers that are typical of an invasive metastatic extracellular matrix (ECM) phenotype. Our data highlight the importance of understanding the functional roles of CD44 in shaping the ECM, including Col1 fibers, in choline metabolism, vascularization, and in invasion and metastasis. CD44 also presents an important target in triple negative breast cancer (TNBC) where chemotherapy is currently the only recourse for treatment. Preliminary studies using antibody based phototherapy (PT) to eliminate CD44 expressing cancer cells showed a dramatic reduction of tumor volume in a CD44 expressing triple negative MDA-MB-231 human breast cancer xenograft. These data have shaped the new directions of this renewal application. In Aim 1 we will understand the role of CD44 in modifying Col1 fiber patterns, attracting CAFs, modifying choline metabolism, altering vascularization and hypoxia, and decreasing invasion and metastasis in MDA-MB-231, SUM-159 and SUM-149 TNBC xenografts expressing short hair pin RNA (shRNA) to downregulate CD44. The applications of molecular and functional imaging to expand our understanding of CD44 as a molecule that is known to influence survival and treatment outcome are essential for improving treatment strategies for TNBC as well as other cancers where CD44 is associated with a stem cell like phenotype. In Aim 2 we will develop and optimize antibody based PT of CD44 expressing cancer cells and CAFs in MDA-MB-231, SUM-159 and SUM-149 TNBC xenografts and the MMTV- PyV MT GEMM (genetically engineered mouse model) of breast cancer, and determine the effect on tumor growth, metastasis, CAFs, Col1 fiber patterns, choline metabolism and vascularization. As part of this aim we will develop catheter and endoscopy strategies to detect and treat CD44 expressing tumors for future human applications of PT. Despite an initial response to chemotherapy, TNBCs relapse, display refractory drug- resistance, and metastasize earlier than other subtypes. There is an urgent unmet need for effective precision medicine of TNBC. PT using a phthalocyanine dye such as IR700 conjugated to CD44 antibody provides a safe and specific strategy to eliminate CD44 expressing cancer cells and CAFs with a clear translational path.